Fecal Bile Acids in Alcoholic Hepatitis

Data setup and objectives

Cohort demographics

Stool data

	level	HDC	MAH	SAH	p
n		20	8	21
age (mean (SD))		46.39 (13.63)	54.33 (7.09)	43.21 (11.36)	0.089
GENDER (%)	F	8 (40.0)	1 (12.5)	11 ( 52.4)	0.148
	M	12 (60.0)	7 (87.5)	10 ( 47.6)
BMI (mean (SD))		29.56 (7.51)	26.92 (2.60)	28.35 (6.09)	0.673
race (%)	Black or African American	1 ( 5.0)	1 (12.5)	0 ( 0.0)	0.421
	More than one race	1 ( 5.0)	0 ( 0.0)	0 ( 0.0)
	White	18 (90.0)	7 (87.5)	21 (100.0)
HEMOGLOBIN (mean (SD))		13.21 (1.43)	9.84 (1.82)	10.36 (1.90)	<0.001
WBC (mean (SD))		6.75 (2.69)	13.74 (8.70)	16.56 (12.83)	0.008
PLATELET (mean (SD))		265.89 (52.83)	207.60 (175.51)	155.00 (88.64)	0.002
CREATININE (mean (SD))		0.89 (0.35)	0.67 (0.31)	1.09 (0.71)	0.257
AST (mean (SD))		25.00 (8.27)	158.20 (91.16)	114.50 (44.97)	<0.001
ALT (mean (SD))		25.11 (12.36)	63.20 (42.86)	40.40 (21.07)	0.002
TOTAL_BILIRUBIN (mean (SD))		0.44 (0.27)	7.06 (3.72)	19.57 (11.36)	<0.001
ALBUMIN (mean (SD))		4.06 (0.52)	2.66 (0.15)	2.82 (0.52)	<0.001
INR (mean (SD))		1.00 (0.15)	1.37 (0.20)	2.07 (0.40)	<0.001
MELD_SCORE (mean (SD))		7.37 (1.74)	14.62 (3.85)	26.81 (5.32)	<0.001
CHILDPUGH_SCORE (mean (SD))		5.21 (0.43)	9.00 (0.71)	10.45 (1.64)	<0.001
DF_SCORE (mean (SD))		12.54 (8.79)	35.95 (7.00)	81.33 (31.75)	<0.001

Plasma data

	level	HC	HDC	MAH	SAH	p
n		20	12	11	16
age (mean (SD))		39.85 (11.32)	36.50 (13.45)	51.19 (9.57)	48.85 (6.76)	0.008
GENDER (%)	F	10 (50.0)	4 (33.3)	4 (36.4)	6 (37.5)	0.767
	M	10 (50.0)	8 (66.7)	7 (63.6)	10 (62.5)
BMI (mean (SD))		26.81 (6.46)	25.08 (6.73)	25.02 (3.82)	27.67 (5.95)	0.594
race (%)	Asian	1 ( 5.0)	0 ( 0.0)	0 ( 0.0)	0 ( 0.0)	0.187
	Black or African American	0 ( 0.0)	2 (16.7)	3 (27.3)	1 ( 6.2)
	More than one race	0 ( 0.0)	1 ( 8.3)	0 ( 0.0)	0 ( 0.0)
	White	19 (95.0)	9 (75.0)	8 (72.7)	15 (93.8)
HEMOGLOBIN (mean (SD))		13.97 (1.61)	11.86 (2.59)	10.00 (2.34)	9.78 (2.39)	<0.001
WBC (mean (SD))		7.18 (1.66)	9.00 (3.34)	9.89 (6.58)	10.05 (5.12)	0.451
PLATELET (mean (SD))		258.33 (89.63)	287.36 (146.47)	145.90 (83.98)	154.94 (88.92)	0.003
CREATININE (mean (SD))		0.89 (0.16)	0.83 (0.41)	0.74 (0.20)	0.93 (0.54)	0.652
AST (mean (SD))		83.33 (171.40)	28.27 (11.91)	141.70 (97.87)	109.56 (29.47)	0.036
ALT (mean (SD))		36.89 (34.44)	21.18 (9.87)	44.30 (23.37)	49.31 (55.93)	0.308
TOTAL_BILIRUBIN (mean (SD))		0.62 (0.49)	0.55 (0.47)	4.88 (2.60)	17.16 (9.64)	<0.001
ALBUMIN (mean (SD))		4.36 (0.24)	3.55 (0.63)	2.74 (0.51)	2.78 (0.61)	<0.001
INR (mean (SD))		NaN (NA)	1.13 (0.23)	1.50 (0.41)	2.04 (0.45)	<0.001
MELD_SCORE (mean (SD))		NaN (NA)	8.50 (3.50)	15.73 (4.22)	25.38 (3.93)	<0.001
CHILDPUGH_SCORE (mean (SD))		NaN (NA)	5.70 (0.67)	8.70 (1.70)	10.31 (1.74)	<0.001
DF_SCORE (mean (SD))		NaN (NA)	-2.60 (11.66)	22.04 (20.72)	59.31 (21.56)	<0.001

Significant metabolites

Primary and secondary bile acids in stool

[1] 1
[1] 2
[1] 3
[1] 4
[1] 5
[1] 6
[1] 7
[1] 8
[1] 9
[1] 10
[1] 11
[1] 12
[1] 13
[1] 14
[1] 15
[1] 16
[1] 17
[1] 18
[1] 19
[1] 20
[1] 21
[1] 22
[1] 23
[1] 24
[1] 25
[1] 26
[1] 27
[1] 28
[1] 29
[1] 30
[1] 31
[1] 32
[1] 33

Plot only those metabolites that had at least one comparison with a significant q-value.

	comparison	BIOCHEMICAL	.y.	group1	group2	n1	n2	statistic	df	p	p.adj	p.adj.signif	q.value	q.value.sign	FoldChange	log2FoldChange	diffexpressed
9	AH_HDC	dehydrolithocholate	log10_value	HDC	AH	20	29	5.639	42.385	0.000	0.000	****	0.000	TRUE	0.119	-3.070	DOWN
10	AH_HDC	deoxycholate	log10_value	HDC	AH	20	29	7.129	46.979	0.000	0.000	****	0.000	TRUE	0.070	-3.843	DOWN
12	AH_HDC	glycochenodeoxycholate glucuronide (1)	log10_value	HDC	AH	20	29	-2.784	28.467	0.009	0.049		0.032	TRUE	14.504	3.858	UP
13	AH_HDC	glycochenodeoxycholate sulfate	log10_value	HDC	AH	20	29	-2.882	33.313	0.007	0.042		0.027	TRUE	309.187	8.272	UP
15	AH_HDC	glycocholate glucuronide (1)	log10_value	HDC	AH	20	29	-2.374	28.000	0.025	0.119	ns	0.077	TRUE	8.515	3.090	NO
16	AH_HDC	glycocholate sulfate	log10_value	HDC	AH	20	29	-2.328	28.000	0.027	0.119	ns	0.077	TRUE	24.512	4.615	NO
17	AH_HDC	glycocholenate sulfate*	log10_value	HDC	AH	20	29	-3.061	45.261	0.004	0.029		0.019	TRUE	13.998	3.807	UP
18	AH_HDC	glycodeoxycholate	log10_value	HDC	AH	20	29	6.127	23.891	0.000	0.000	****	0.000	TRUE	0.037	-4.765	DOWN
20	AH_HDC	glycolithocholate	log10_value	HDC	AH	20	29	2.391	19.235	0.027	0.119	ns	0.077	TRUE	0.466	-1.101	NO
23	AH_HDC	isoursodeoxycholate	log10_value	HDC	AH	20	29	4.089	46.887	0.000	0.002	**	0.001	TRUE	0.254	-1.974	DOWN
24	AH_HDC	lithocholate	log10_value	HDC	AH	20	29	7.480	41.242	0.000	0.000	****	0.000	TRUE	0.168	-2.577	DOWN
25	AH_HDC	taurochenodeoxycholate	log10_value	HDC	AH	20	29	-3.141	46.354	0.003	0.028		0.018	TRUE	7.829	2.969	UP
27	AH_HDC	taurocholenate sulfate	log10_value	HDC	AH	20	29	-3.720	45.640	0.001	0.006	**	0.004	TRUE	21.308	4.413	UP
28	AH_HDC	taurodeoxycholate	log10_value	HDC	AH	20	29	2.966	23.718	0.007	0.042		0.027	TRUE	0.115	-3.114	DOWN
31	AH_HDC	ursocholate	log10_value	HDC	AH	20	29	2.216	46.868	0.032	0.132	ns	0.085	TRUE	0.382	-1.388	NO
32	AH_HDC	ursodeoxycholate	log10_value	HDC	AH	20	29	2.988	46.904	0.004	0.029		0.019	TRUE	0.352	-1.506	DOWN
42	SAH_MAH	dehydrolithocholate	log10_value	MAH	SAH	8	21	0.688	13.199	0.503	0.745	ns	0.481	FALSE	2.370	1.245	NO
43	SAH_MAH	deoxycholate	log10_value	MAH	SAH	8	21	1.595	11.056	0.139	0.417	ns	0.269	FALSE	0.153	-2.709	NO
45	SAH_MAH	glycochenodeoxycholate glucuronide (1)	log10_value	MAH	SAH	8	21	-0.345	13.031	0.736	0.836	ns	0.540	FALSE	2.305	1.205	NO
46	SAH_MAH	glycochenodeoxycholate sulfate	log10_value	MAH	SAH	8	21	0.399	11.736	0.697	0.836	ns	0.540	FALSE	4.014	2.005	NO
48	SAH_MAH	glycocholate glucuronide (1)	log10_value	MAH	SAH	8	21	-0.122	14.571	0.905	0.963	ns	0.622	FALSE	2.586	1.371	NO
49	SAH_MAH	glycocholate sulfate	log10_value	MAH	SAH	8	21	0.002	10.978	0.998	0.998	ns	0.645	FALSE	1.388	0.473	NO
50	SAH_MAH	glycocholenate sulfate*	log10_value	MAH	SAH	8	21	0.332	10.153	0.747	0.836	ns	0.540	FALSE	0.324	-1.625	NO
51	SAH_MAH	glycodeoxycholate	log10_value	MAH	SAH	8	21	1.425	7.761	0.193	0.464	ns	0.300	FALSE	0.306	-1.709	NO
53	SAH_MAH	glycolithocholate	log10_value	MAH	SAH	8	21	-1.196	20.000	0.246	0.494	ns	0.319	FALSE	1.025	0.035	NO
56	SAH_MAH	isoursodeoxycholate	log10_value	MAH	SAH	8	21	1.528	11.987	0.153	0.439	ns	0.284	FALSE	0.449	-1.156	NO
57	SAH_MAH	lithocholate	log10_value	MAH	SAH	8	21	1.015	11.913	0.330	0.605	ns	0.391	FALSE	0.577	-0.793	NO
58	SAH_MAH	taurochenodeoxycholate	log10_value	MAH	SAH	8	21	-0.681	12.788	0.508	0.745	ns	0.481	FALSE	2.964	1.567	NO
60	SAH_MAH	taurocholenate sulfate	log10_value	MAH	SAH	8	21	-0.067	9.459	0.948	0.978	ns	0.632	FALSE	0.211	-2.242	NO
61	SAH_MAH	taurodeoxycholate	log10_value	MAH	SAH	8	21	0.906	8.867	0.389	0.658	ns	0.425	FALSE	0.582	-0.782	NO
64	SAH_MAH	ursocholate	log10_value	MAH	SAH	8	21	1.921	12.045	0.079	0.274	ns	0.177	FALSE	0.260	-1.945	NO
65	SAH_MAH	ursodeoxycholate	log10_value	MAH	SAH	8	21	1.365	11.010	0.200	0.464	ns	0.300	FALSE	0.361	-1.472	NO

Kruskal-Wallis tests on all metabolites to identify which are significant

	ID	BIOCHEMICAL	KW.pvalue	q.value	q.value.sign	Primary_Secondary	Conj_Unconj	Conj_type	Comp12_OH	Comp_12-OH_Fig1_2	Dihydroxy_Trihydroxy	Comp_7-dehydroxy	Comp_7-dehydro_Fig_1_2	Keto	Epimer
6	M_2715	taurochenodeoxycholate	0.010	0.011	TRUE	P	C	T	n12	2	2	n7	1	n	n
10	M_2727	glycocholate glucuronide (1)	0.041	0.028	TRUE	P	C	G/Glu	12	1	2	n7	1	n	n
11	M_2728	glycocholate sulfate	0.039	0.027	TRUE	P	C	G/S	12	1	2	n7	1	n	n
12	M_2729	deoxycholate	0.000	0.000	TRUE	S	UC	0	12	1	2	7	2	n	n
13	M_2731	glycodeoxycholate	0.000	0.000	TRUE	S	C	G	12	1	2	7	2	n	n
14	M_2732	taurodeoxycholate	0.002	0.004	TRUE	S	C	T	12	1	2	7	2	n	n
15	M_2734	lithocholate	0.000	0.000	TRUE	S	UC	0	n12	2	1	7	2	n	n
16	M_2736	glycolithocholate	0.013	0.012	TRUE	S	C	G	n12	2	1	7	2	n	n
19	M_2741	ursodeoxycholate	0.010	0.011	TRUE	S	UC	0	n12	2	2 (1b)	n7 (b)	N/A	n	y
20	M_2742	isoursodeoxycholate	0.002	0.004	TRUE	S	UC	0	n12	2	2 (2b)	n7 (b)	N/A	n	y
23	M_2745	dehydrolithocholate	0.000	0.001	TRUE	S	UC	0	n12	2	0	7	2	y	n
28	M_2760	glycocholenate sulfate*	0.033	0.024	TRUE	O	C	G/S	12	1	2	n7	1	n	n
29	M_2761	taurocholenate sulfate	0.006	0.007	TRUE	O	C	T/S	12	1	2	n7	1	n	n
32	M_2767	ursodeoxycholate sulfate (1)	0.034	0.024	TRUE	S	C	S	n12	2	1 (1b)	n7 (b)	N/A	n	y
33	M_2770	ursocholate	0.014	0.013	TRUE	S	UC	0	12	1	3 (2b)	n7 (b)	N/A	n	y

Primary and secondary bile acids in plasma

Note: for the glycocholate sulfate, the healthy and heavy drinking controls have the identical imputed values. Therefore one group is removed from the test. Also, be careful when comparing t-test with control

Plot only those metabolites that had at least one comparison with a significant q-value.

	comparison	BIOCHEMICAL	.y.	group1	group2	n1	n2	statistic	df	p	p.adj	p.adj.signif	q.value	q.value.sign	FoldChange	log2FoldChange	diffexpressed
3	AH_HDC	deoxycholate	log10_value	HDC	AH	12	27	2.270	25.802	0.032	0.090	ns	0.086	TRUE	0.634	-0.657	DOWN
4	AH_HDC	glycochenodeoxycholate	log10_value	HDC	AH	12	27	-8.720	16.539	0.000	0.000	****	0.000	TRUE	15.846	3.986	UP
5	AH_HDC	glycochenodeoxycholate glucuronide (1)	log10_value	HDC	AH	12	27	-6.402	21.685	0.000	0.000	****	0.000	TRUE	8.617	3.107	UP
6	AH_HDC	glycochenodeoxycholate sulfate	log10_value	HDC	AH	12	27	-6.785	15.541	0.000	0.000	****	0.000	TRUE	14.966	3.904	UP
7	AH_HDC	glycocholate	log10_value	HDC	AH	12	27	-10.135	20.444	0.000	0.000	****	0.000	TRUE	17.249	4.108	UP
8	AH_HDC	glycocholate sulfate	log10_value	HDC	AH	12	27	-6.106	26.000	0.000	0.000	****	0.000	TRUE	15.651	3.968	UP
9	AH_HDC	glycocholenate sulfate*	log10_value	HDC	AH	12	27	-4.551	21.249	0.000	0.001	***	0.001	TRUE	3.258	1.704	UP
10	AH_HDC	glycodeoxycholate	log10_value	HDC	AH	12	27	2.849	25.443	0.009	0.034		0.033	TRUE	3.053	1.610	UP
12	AH_HDC	glycolithocholate	log10_value	HDC	AH	12	27	-2.567	36.993	0.014	0.048		0.046	TRUE	15.606	3.964	UP
13	AH_HDC	glycolithocholate sulfate*	log10_value	HDC	AH	12	27	-2.884	13.729	0.012	0.043		0.041	TRUE	4.061	2.022	UP
14	AH_HDC	glycoursodeoxycholate	log10_value	HDC	AH	12	27	-2.417	24.874	0.023	0.075	ns	0.071	TRUE	8.129	3.023	UP
15	AH_HDC	isoursodeoxycholate	log10_value	HDC	AH	12	27	3.736	18.583	0.001	0.005	**	0.004	TRUE	0.174	-2.520	DOWN
16	AH_HDC	taurochenodeoxycholate	log10_value	HDC	AH	12	27	-12.057	14.596	0.000	0.000	****	0.000	TRUE	93.188	6.542	UP
17	AH_HDC	taurocholate	log10_value	HDC	AH	12	27	-9.910	19.373	0.000	0.000	****	0.000	TRUE	127.817	6.998	UP
18	AH_HDC	taurocholenate sulfate	log10_value	HDC	AH	12	27	-7.315	14.536	0.000	0.000	****	0.000	TRUE	11.732	3.552	UP
19	AH_HDC	taurodeoxycholate	log10_value	HDC	AH	12	27	2.823	24.406	0.009	0.034		0.033	TRUE	5.281	2.401	UP
20	AH_HDC	taurolithocholate 3-sulfate	log10_value	HDC	AH	12	27	-5.973	14.333	0.000	0.000	***	0.000	TRUE	15.564	3.960	UP
21	AH_HDC	tauroursodeoxycholate	log10_value	HDC	AH	12	27	-6.853	19.087	0.000	0.000	****	0.000	TRUE	130.767	7.031	UP
25	HDC_HC	deoxycholate	log10_value	HC	HDC	20	12	2.123	21.283	0.046	0.115	ns	0.110	FALSE	0.587	-0.769	NO
26	HDC_HC	glycochenodeoxycholate	log10_value	HC	HDC	20	12	-3.774	20.920	0.001	0.005	**	0.004	TRUE	4.247	2.087	UP
27	HDC_HC	glycochenodeoxycholate glucuronide (1)	log10_value	HC	HDC	20	12	-1.703	26.319	0.100	0.224	ns	0.214	FALSE	1.526	0.610	NO
28	HDC_HC	glycochenodeoxycholate sulfate	log10_value	HC	HDC	20	12	-3.132	19.137	0.005	0.022		0.021	TRUE	6.320	2.660	UP
29	HDC_HC	glycocholate	log10_value	HC	HDC	20	12	-4.637	27.874	0.000	0.000	***	0.000	TRUE	3.110	1.637	UP
30	HDC_HC	glycocholenate sulfate*	log10_value	HC	HDC	20	12	-1.891	15.506	0.078	0.181	ns	0.173	FALSE	1.741	0.800	NO
31	HDC_HC	glycodeoxycholate	log10_value	HC	HDC	20	12	-0.041	18.315	0.968	0.985	ns	0.940	FALSE	1.293	0.370	NO
33	HDC_HC	glycolithocholate	log10_value	HC	HDC	20	12	0.329	25.225	0.745	0.866	ns	0.826	FALSE	0.827	-0.274	NO
34	HDC_HC	glycolithocholate sulfate*	log10_value	HC	HDC	20	12	0.730	12.246	0.479	0.708	ns	0.675	FALSE	1.642	0.716	NO
35	HDC_HC	glycoursodeoxycholate	log10_value	HC	HDC	20	12	-2.311	20.022	0.032	0.090	ns	0.086	TRUE	3.007	1.588	UP
36	HDC_HC	isoursodeoxycholate	log10_value	HC	HDC	20	12	1.558	21.088	0.134	0.272	ns	0.260	FALSE	0.369	-1.437	NO
37	HDC_HC	taurochenodeoxycholate	log10_value	HC	HDC	20	12	-2.238	19.338	0.037	0.096	ns	0.092	TRUE	2.823	1.497	UP
38	HDC_HC	taurocholate	log10_value	HC	HDC	20	12	-2.331	22.045	0.029	0.090	ns	0.086	TRUE	2.911	1.541	UP
39	HDC_HC	taurocholenate sulfate	log10_value	HC	HDC	20	12	-0.007	16.079	0.994	0.994	ns	0.948	FALSE	1.812	0.858	NO
40	HDC_HC	taurodeoxycholate	log10_value	HC	HDC	20	12	0.378	16.953	0.710	0.866	ns	0.826	FALSE	1.269	0.344	NO
41	HDC_HC	taurolithocholate 3-sulfate	log10_value	HC	HDC	20	12	0.830	14.507	0.420	0.702	ns	0.670	FALSE	1.571	0.652	NO
42	HDC_HC	tauroursodeoxycholate	log10_value	HC	HDC	20	12	-2.319	15.173	0.035	0.095	ns	0.090	TRUE	5.382	2.428	UP
46	SAH_MAH	deoxycholate	log10_value	MAH	SAH	11	16	0.745	15.237	0.468	0.708	ns	0.675	FALSE	0.376	-1.410	NO
47	SAH_MAH	glycochenodeoxycholate	log10_value	MAH	SAH	11	16	0.163	19.600	0.872	0.977	ns	0.932	FALSE	0.957	-0.064	NO
48	SAH_MAH	glycochenodeoxycholate glucuronide (1)	log10_value	MAH	SAH	11	16	0.050	18.412	0.961	0.985	ns	0.940	FALSE	0.708	-0.499	NO
49	SAH_MAH	glycochenodeoxycholate sulfate	log10_value	MAH	SAH	11	16	0.464	20.345	0.648	0.826	ns	0.788	FALSE	0.762	-0.392	NO
50	SAH_MAH	glycocholate	log10_value	MAH	SAH	11	16	0.552	16.172	0.588	0.764	ns	0.729	FALSE	0.712	-0.490	NO
51	SAH_MAH	glycocholate sulfate	log10_value	MAH	SAH	11	16	0.651	20.915	0.522	0.719	ns	0.686	FALSE	0.525	-0.930	NO
52	SAH_MAH	glycocholenate sulfate*	log10_value	MAH	SAH	11	16	0.677	23.025	0.505	0.714	ns	0.681	FALSE	0.963	-0.055	NO
53	SAH_MAH	glycodeoxycholate	log10_value	MAH	SAH	11	16	1.487	10.000	0.168	0.321	ns	0.306	FALSE	0.011	-6.519	NO
55	SAH_MAH	glycolithocholate	log10_value	MAH	SAH	11	16	-0.233	15.541	0.819	0.934	ns	0.891	FALSE	0.371	-1.431	NO
56	SAH_MAH	glycolithocholate sulfate*	log10_value	MAH	SAH	11	16	0.095	13.664	0.926	0.985	ns	0.940	FALSE	0.397	-1.333	NO
57	SAH_MAH	glycoursodeoxycholate	log10_value	MAH	SAH	11	16	0.637	22.843	0.531	0.719	ns	0.686	FALSE	4.006	2.002	NO
58	SAH_MAH	isoursodeoxycholate	log10_value	MAH	SAH	11	16	1.935	21.252	0.066	0.159	ns	0.152	FALSE	0.502	-0.993	NO
59	SAH_MAH	taurochenodeoxycholate	log10_value	MAH	SAH	11	16	-1.666	16.862	0.114	0.239	ns	0.228	FALSE	1.589	0.668	NO
60	SAH_MAH	taurocholate	log10_value	MAH	SAH	11	16	-0.806	21.233	0.429	0.702	ns	0.670	FALSE	1.629	0.704	NO
61	SAH_MAH	taurocholenate sulfate	log10_value	MAH	SAH	11	16	-0.131	23.542	0.897	0.985	ns	0.940	FALSE	1.419	0.505	NO
62	SAH_MAH	taurodeoxycholate	log10_value	MAH	SAH	11	16	1.491	10.000	0.167	0.321	ns	0.306	FALSE	0.003	-8.582	NO
63	SAH_MAH	taurolithocholate 3-sulfate	log10_value	MAH	SAH	11	16	-0.739	17.094	0.470	0.708	ns	0.675	FALSE	0.901	-0.150	NO
64	SAH_MAH	tauroursodeoxycholate	log10_value	MAH	SAH	11	16	-0.683	23.997	0.501	0.714	ns	0.681	FALSE	9.394	3.232	NO

Correlation of significant BA with taxa

HDC and AH correlations

MAH and SAH correlations

SAH correlations

Taxa sensitive and resistant to bile acids

Taxa linked to bile acids

Conjugation group heatmaps

Stool total primary and total secondary heatmaps

Boxplots of the total primary concentration in each sample

T-tests on the log scale: primary

total.primary.lm_stool <- lm(log10(value) ~ Class, data = subset(total.primary_stool, 
    Class != "AH" & Primary_Secondary == "P"))
anova(total.primary.lm_stool)

Analysis of Variance Table

Response: log10(value)
          Df  Sum Sq Mean Sq F value Pr(>F)
Class      2  0.9575 0.47874  1.0076  0.373
Residuals 46 21.8554 0.47512               

total.primary.lsm <- lsmeans::lsmeans(total.primary.lm_stool, pairwise ~ Class)
total.primary.lsm

$lsmeans
 Class lsmean    SE df lower.CL upper.CL
 HDC     1.06 0.154 46    0.751     1.37
 MAH     1.38 0.244 46    0.887     1.87
 SAH     1.33 0.150 46    1.028     1.63

Results are given on the log10 (not the response) scale. 
Confidence level used: 0.95 

$contrasts
 contrast  estimate    SE df t.ratio p.value
 HDC - MAH  -0.3164 0.288 46 -1.097  0.5205 
 HDC - SAH  -0.2696 0.215 46 -1.252  0.4294 
 MAH - SAH   0.0469 0.286 46  0.164  0.9854 

Note: contrasts are still on the log10 scale 
P value adjustment: tukey method for comparing a family of 3 estimates 

T-tests on the log scale: secondary

total.secondary.lm_stool <- lm(log10(value) ~ Class, data = subset(total.primary_stool, 
    Class != "AH" & Primary_Secondary == "S"))
anova(total.secondary.lm_stool)

Analysis of Variance Table

Response: log10(value)
          Df Sum Sq Mean Sq F value Pr(>F)  
Class      2  2.750 1.37498  4.2642   0.02 *
Residuals 46 14.833 0.32245                 
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

total.secondary.lsm <- lsmeans::lsmeans(total.secondary.lm_stool, pairwise ~ Class)
total.secondary.lsm

$lsmeans
 Class lsmean    SE df lower.CL upper.CL
 HDC     1.72 0.127 46    1.460     1.97
 MAH     1.61 0.201 46    1.211     2.02
 SAH     1.21 0.124 46    0.964     1.46

Results are given on the log10 (not the response) scale. 
Confidence level used: 0.95 

$contrasts
 contrast  estimate    SE df t.ratio p.value
 HDC - MAH    0.101 0.238 46 0.424   0.9057 
 HDC - SAH    0.502 0.177 46 2.832   0.0184 
 MAH - SAH    0.402 0.236 46 1.702   0.2153 

Note: contrasts are still on the log10 scale 
P value adjustment: tukey method for comparing a family of 3 estimates 

Conjugated unconjugated

Boxplots of the total conjugated concentration in each sample

T-tests on the log scale: conjugated

total.conj.lm_stool <- lm(log10(value) ~ Class, data = subset(total.conj_stool, Class != 
    "AH" & Conj_Unconj == "C"))
anova(total.conj.lm_stool)

Analysis of Variance Table

Response: log10(value)
          Df  Sum Sq Mean Sq F value Pr(>F)
Class      2  0.3214 0.16068  0.3541 0.7037
Residuals 46 20.8722 0.45374               

T-tests on the log scale: unconjugated

total.unconj.lm_stool <- lm(log10(value) ~ Class, data = subset(total.conj_stool, 
    Class != "AH" & Conj_Unconj == "UC"))
anova(total.unconj.lm_stool)

Analysis of Variance Table

Response: log10(value)
          Df  Sum Sq Mean Sq F value  Pr(>F)  
Class      2  2.6863 1.34313   2.618 0.08379 .
Residuals 46 23.5998 0.51304                  
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

total.unconj.lsm <- lsmeans::lsmeans(total.unconj.lm_stool, pairwise ~ Class)
total.unconj.lsm

$lsmeans
 Class lsmean    SE df lower.CL upper.CL
 HDC    1.496 0.160 46    1.174     1.82
 MAH    1.304 0.253 46    0.795     1.81
 SAH    0.987 0.156 46    0.672     1.30

Results are given on the log10 (not the response) scale. 
Confidence level used: 0.95 

$contrasts
 contrast  estimate    SE df t.ratio p.value
 HDC - MAH    0.192 0.300 46 0.640   0.7991 
 HDC - SAH    0.509 0.224 46 2.275   0.0696 
 MAH - SAH    0.317 0.298 46 1.067   0.5394 

Note: contrasts are still on the log10 scale 
P value adjustment: tukey method for comparing a family of 3 estimates 

Plasma total primary and total secondary heatmaps

Boxplots of the total secondary concentration in each sample

T-tests on the log scale:primary

total.primary.lm_plasma <- lm(log10(value) ~ Class, data = subset(total.primary_plasma, 
    Class != "AH" & Primary_Secondary == "P"))
anova(total.primary.lm_plasma)

Analysis of Variance Table

Response: log10(value)
          Df  Sum Sq Mean Sq F value    Pr(>F)    
Class      3 28.9838  9.6613  81.042 < 2.2e-16 ***
Residuals 55  6.5567  0.1192                      
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

total.primary.lsm <- lsmeans::lsmeans(total.primary.lm_plasma, pairwise ~ Class)
total.primary.lsm

$lsmeans
 Class lsmean     SE df lower.CL upper.CL
 HC     0.505 0.0772 55    0.350    0.660
 HDC    0.721 0.0997 55    0.521    0.921
 MAH    1.918 0.1041 55    1.709    2.127
 SAH    2.026 0.0863 55    1.854    2.199

Results are given on the log10 (not the response) scale. 
Confidence level used: 0.95 

$contrasts
 contrast  estimate    SE df t.ratio p.value
 HC - HDC    -0.216 0.126 55  -1.715 0.3259 
 HC - MAH    -1.413 0.130 55 -10.904 <.0001 
 HC - SAH    -1.522 0.116 55 -13.140 <.0001 
 HDC - MAH   -1.197 0.144 55  -8.306 <.0001 
 HDC - SAH   -1.305 0.132 55  -9.901 <.0001 
 MAH - SAH   -0.108 0.135 55  -0.801 0.8534 

Note: contrasts are still on the log10 scale 
P value adjustment: tukey method for comparing a family of 4 estimates 

T-tests on the log scale: secondary

total.secondary.lm_plasma <- lm(log10(value) ~ Class, data = subset(total.primary_plasma, 
    Class != "AH" & Primary_Secondary == "S"))
anova(total.secondary.lm_plasma)

Analysis of Variance Table

Response: log10(value)
          Df  Sum Sq Mean Sq F value Pr(>F)
Class      3  1.2743 0.42475  1.7944  0.159
Residuals 55 13.0188 0.23671               

total.secondary.lsm <- lsmeans::lsmeans(total.secondary.lm_plasma, pairwise ~ Class)
total.secondary.lsm

$lsmeans
 Class lsmean    SE df lower.CL upper.CL
 HC      1.11 0.109 55    0.893     1.33
 HDC     1.05 0.140 55    0.766     1.33
 MAH     1.41 0.147 55    1.116     1.70
 SAH     1.35 0.122 55    1.109     1.60

Results are given on the log10 (not the response) scale. 
Confidence level used: 0.95 

$contrasts
 contrast  estimate    SE df t.ratio p.value
 HC - HDC    0.0636 0.178 55  0.358  0.9841 
 HC - MAH   -0.2992 0.183 55 -1.638  0.3661 
 HC - SAH   -0.2416 0.163 55 -1.481  0.4560 
 HDC - MAH  -0.3628 0.203 55 -1.786  0.2908 
 HDC - SAH  -0.3052 0.186 55 -1.643  0.3638 
 MAH - SAH   0.0576 0.191 55  0.302  0.9903 

Note: contrasts are still on the log10 scale 
P value adjustment: tukey method for comparing a family of 4 estimates 

Conjugated unconjugated

Boxplots of the total conjugated concentration in each sample

T-tests on the log scale: conjugated

# Conj_Unconj == 'C'
total.conj.lm_plasma <- lm(log10(value) ~ Class, data = subset(total.conj_plasma, 
    Class != "AH" & Conj_Unconj == "C"))
anova(total.conj.lm_plasma)

Analysis of Variance Table

Response: log10(value)
          Df  Sum Sq Mean Sq F value    Pr(>F)    
Class      3 23.3016  7.7672   80.08 < 2.2e-16 ***
Residuals 55  5.3346  0.0970                      
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

total.conj.lsm <- lsmeans::lsmeans(total.conj.lm_plasma, pairwise ~ Class)
total.conj.lsm

$lsmeans
 Class lsmean     SE df lower.CL upper.CL
 HC     0.815 0.0696 55    0.675    0.954
 HDC    1.016 0.0899 55    0.835    1.196
 MAH    2.123 0.0939 55    1.935    2.311
 SAH    2.157 0.0779 55    2.001    2.313

Results are given on the log10 (not the response) scale. 
Confidence level used: 0.95 

$contrasts
 contrast  estimate    SE df t.ratio p.value
 HC - HDC    -0.201 0.114 55  -1.764 0.3013 
 HC - MAH    -1.308 0.117 55 -11.189 <.0001 
 HC - SAH    -1.342 0.104 55 -12.848 <.0001 
 HDC - MAH   -1.107 0.130 55  -8.519 <.0001 
 HDC - SAH   -1.141 0.119 55  -9.598 <.0001 
 MAH - SAH   -0.034 0.122 55  -0.279 0.9924 

Note: contrasts are still on the log10 scale 
P value adjustment: tukey method for comparing a family of 4 estimates 

T-tests on the log scale: unconjugated

total.unconj.lm_plasma <- lm(log10(value) ~ Class, data = subset(total.conj_plasma, 
    Class != "AH" & Conj_Unconj == "UC"))
anova(total.unconj.lm_plasma)

Analysis of Variance Table

Response: log10(value)
          Df  Sum Sq Mean Sq F value Pr(>F)
Class      3  0.5114 0.17047  0.7029 0.5544
Residuals 55 13.3401 0.24255               

Precursor: 7-alpha-hydroxy-3-oxo-4-cholestenoate (7-Hoca) 2530

T-tests on the log scale:7-HOCA

HOCA.lm_plasma <- lm(log10(value) ~ Class, data = M_2530_df.m)
anova(HOCA.lm_plasma)

Analysis of Variance Table

Response: log10(value)
          Df Sum Sq Mean Sq F value    Pr(>F)    
Class      3 4.0157 1.33856  21.627 2.214e-09 ***
Residuals 55 3.4041 0.06189                      
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

HOCA.lm_plasma.lsm <- lsmeans::lsmeans(HOCA.lm_plasma, pairwise ~ Class)
HOCA.lm_plasma.lsm

$lsmeans
 Class  lsmean     SE df lower.CL upper.CL
 HC    -0.0394 0.0556 55   -0.151   0.0721
 HDC   -0.2093 0.0718 55   -0.353  -0.0654
 MAH    0.2807 0.0750 55    0.130   0.4310
 SAH    0.4667 0.0622 55    0.342   0.5913

Results are given on the log10 (not the response) scale. 
Confidence level used: 0.95 

$contrasts
 contrast  estimate     SE df t.ratio p.value
 HC - HDC     0.170 0.0908 55  1.871  0.2525 
 HC - MAH    -0.320 0.0934 55 -3.428  0.0062 
 HC - SAH    -0.506 0.0834 55 -6.064  <.0001 
 HDC - MAH   -0.490 0.1038 55 -4.719  0.0001 
 HDC - SAH   -0.676 0.0950 55 -7.115  <.0001 
 MAH - SAH   -0.186 0.0974 55 -1.908  0.2365 

Note: contrasts are still on the log10 scale 
P value adjustment: tukey method for comparing a family of 4 estimates 

Figure 1

put 7-hoca in the middel as separator

Figure 2: significant metabolites in healthy to heavy drinking comparison

Figure 5: Correlation plots

Stool Random forest models for prediction of AH and disease severity

AH prediction model in stool

Call:
 randomForest(x = x, y = y, ntree = 501, mtry = param$mtry, importance = TRUE) 
               Type of random forest: classification
                     Number of trees: 501
No. of variables tried at each split: 5

        OOB estimate of  error rate: 14.29%
Confusion matrix:
    AH HDC class.error
AH  25   4    0.137931
HDC  3  17    0.150000

Disease severity in stool

Call:
 randomForest(x = x, y = y, ntree = 501, mtry = param$mtry, importance = TRUE) 
               Type of random forest: classification
                     Number of trees: 501
No. of variables tried at each split: 6

        OOB estimate of  error rate: 34.48%
Confusion matrix:
    MAH SAH class.error
MAH   1   7   0.8750000
SAH   3  18   0.1428571

Plasma Random forest models for prediction of AH and disease severity

AH prediction model in plasma

Call:
 randomForest(x = x, y = y, ntree = 501, mtry = param$mtry, importance = TRUE) 
               Type of random forest: classification
                     Number of trees: 501
No. of variables tried at each split: 4

        OOB estimate of  error rate: 2.56%
Confusion matrix:
    AH HDC class.error
AH  26   1  0.03703704
HDC  0  12  0.00000000

Disease severity in plasma

Call:
 randomForest(x = x, y = y, ntree = 501, mtry = param$mtry, importance = TRUE) 
               Type of random forest: classification
                     Number of trees: 501
No. of variables tried at each split: 5

        OOB estimate of  error rate: 44.44%
Confusion matrix:
    MAH SAH class.error
MAH   3   8   0.7272727
SAH   4  12   0.2500000

Figure 4

Supplementary figure 1

Prediction of disease severity

MELD score univariate prediction

Stool metabolites univariate meld prediction

BIOCHEMICAL	SUPER_PATHWAY	SUB_PATHWAY	coeff_AH	p_val_AH	R_sq_AH	coeff_Sev	p_val_Sev	R_sq_Sev
cholate	Lipid	Primary Bile Acid Metabolism	-1.400	0.359	0.018	-1.947	0.238	0.051
glycocholate	Lipid	Primary Bile Acid Metabolism	-0.101	0.948	0.000	-0.413	0.765	0.003
taurocholate	Lipid	Primary Bile Acid Metabolism	1.177	0.451	0.012	0.460	0.738	0.004
chenodeoxycholate	Lipid	Primary Bile Acid Metabolism	0.244	0.875	0.001	-0.732	0.631	0.009
glycochenodeoxycholate	Lipid	Primary Bile Acid Metabolism	1.265	0.460	0.012	-0.086	0.958	0.000
taurochenodeoxycholate	Lipid	Primary Bile Acid Metabolism	4.498	0.006	0.151	1.654	0.320	0.037
cholate sulfate	Lipid	Primary Bile Acid Metabolism	-3.895	0.083	0.064	-2.937	0.242	0.050
glycochenodeoxycholate glucuronide (1)	Lipid	Primary Bile Acid Metabolism	4.613	0.112	0.054	-0.471	0.842	0.002
glycochenodeoxycholate sulfate	Lipid	Primary Bile Acid Metabolism	1.821	0.244	0.029	-1.110	0.388	0.028
glycocholate glucuronide (1)	Lipid	Primary Bile Acid Metabolism	3.394	0.321	0.021	-1.833	0.498	0.017
glycocholate sulfate	Lipid	Primary Bile Acid Metabolism	2.763	0.315	0.022	-1.348	0.534	0.014
deoxycholate	Lipid	Secondary Bile Acid Metabolism	-3.619	0.000	0.393	-0.851	0.402	0.026
glycodeoxycholate	Lipid	Secondary Bile Acid Metabolism	-8.304	0.000	0.429	-6.982	0.102	0.096
taurodeoxycholate	Lipid	Secondary Bile Acid Metabolism	-7.870	0.004	0.167	-1.701	0.748	0.004
lithocholate	Lipid	Secondary Bile Acid Metabolism	-4.154	0.000	0.384	-0.954	0.384	0.028
glycolithocholate	Lipid	Secondary Bile Acid Metabolism	-14.981	0.021	0.110	3.238	0.947	0.000
glycolithocholate sulfate*	Lipid	Secondary Bile Acid Metabolism	-1.496	0.410	0.015	-2.312	0.165	0.070
taurolithocholate 3-sulfate	Lipid	Secondary Bile Acid Metabolism	0.066	0.976	0.000	-2.273	0.227	0.054
ursodeoxycholate	Lipid	Secondary Bile Acid Metabolism	-3.690	0.005	0.158	-1.166	0.370	0.030
isoursodeoxycholate	Lipid	Secondary Bile Acid Metabolism	-5.623	0.001	0.230	-1.689	0.331	0.035
glycoursodeoxycholate	Lipid	Secondary Bile Acid Metabolism	-4.369	0.030	0.099	-3.600	0.073	0.114
tauroursodeoxycholate	Lipid	Secondary Bile Acid Metabolism	-1.014	0.754	0.002	-2.739	0.357	0.032
dehydrolithocholate	Lipid	Secondary Bile Acid Metabolism	-3.969	0.000	0.314	-1.056	0.414	0.025
7,12-diketolithocholate	Lipid	Secondary Bile Acid Metabolism	-1.915	0.542	0.008	1.589	0.655	0.008
7-ketolithocholate	Lipid	Secondary Bile Acid Metabolism	-0.083	0.952	0.000	-1.266	0.384	0.028
3-dehydrocholate	Lipid	Secondary Bile Acid Metabolism	0.462	0.772	0.002	-0.078	0.965	0.000
12-dehydrocholate	Lipid	Secondary Bile Acid Metabolism	-0.847	0.586	0.006	-0.013	0.994	0.000
glycocholenate sulfate*	Lipid	Secondary Bile Acid Metabolism	2.294	0.171	0.040	-1.360	0.365	0.031
taurocholenate sulfate	Lipid	Secondary Bile Acid Metabolism	3.143	0.034	0.095	-0.535	0.703	0.005
7-ketodeoxycholate	Lipid	Secondary Bile Acid Metabolism	-1.911	0.218	0.033	-1.736	0.304	0.039
glycodeoxycholate sulfate	Lipid	Secondary Bile Acid Metabolism	-2.873	0.265	0.027	-2.686	0.227	0.054
ursodeoxycholate sulfate (1)	Lipid	Secondary Bile Acid Metabolism	-4.304	0.002	0.197	-3.670	0.049	0.136
ursocholate	Lipid	Secondary Bile Acid Metabolism	-3.220	0.014	0.125	-1.397	0.257	0.047

Severity prediction model with significant metabolite

Plasma meld prediction

plasma metabolites univariate meld prediction

BIOCHEMICAL	SUPER_PATHWAY	SUB_PATHWAY	coeff_AH	p_val_AH	R_sq_AH	coeff_Sev	p_val_Sev	R_sq_Sev
cholate	Lipid	Primary Bile Acid Metabolism	2.032	0.440	0.017	2.679	0.228	0.058
glycocholate	Lipid	Primary Bile Acid Metabolism	8.663	0.000	0.473	3.293	0.365	0.033
taurocholate	Lipid	Primary Bile Acid Metabolism	4.882	0.000	0.504	3.041	0.141	0.084
chenodeoxycholate	Lipid	Primary Bile Acid Metabolism	4.018	0.169	0.053	3.171	0.256	0.051
glycochenodeoxycholate	Lipid	Primary Bile Acid Metabolism	7.873	0.000	0.416	2.820	0.453	0.023
taurochenodeoxycholate	Lipid	Primary Bile Acid Metabolism	5.625	0.000	0.552	7.968	0.022	0.192
glycochenodeoxycholate glucuronide (1)	Lipid	Primary Bile Acid Metabolism	8.361	0.000	0.349	3.474	0.264	0.050
glycochenodeoxycholate sulfate	Lipid	Primary Bile Acid Metabolism	6.961	0.000	0.437	4.179	0.156	0.079
glycocholate sulfate	Lipid	Primary Bile Acid Metabolism	5.699	0.006	0.198	1.134	0.570	0.013
deoxycholate	Lipid	Secondary Bile Acid Metabolism	-4.306	0.132	0.064	-0.281	0.913	0.000
glycodeoxycholate	Lipid	Secondary Bile Acid Metabolism	-4.256	0.020	0.144	-2.093	0.230	0.057
taurodeoxycholate	Lipid	Secondary Bile Acid Metabolism	-3.312	0.024	0.137	-1.767	0.207	0.063
glycolithocholate	Lipid	Secondary Bile Acid Metabolism	2.895	0.094	0.078	0.813	0.582	0.012
glycolithocholate sulfate*	Lipid	Secondary Bile Acid Metabolism	5.021	0.005	0.206	1.227	0.652	0.008
taurolithocholate 3-sulfate	Lipid	Secondary Bile Acid Metabolism	5.777	0.000	0.439	3.726	0.160	0.077
ursodeoxycholate	Lipid	Secondary Bile Acid Metabolism	-0.280	0.865	0.001	1.719	0.240	0.055
isoursodeoxycholate	Lipid	Secondary Bile Acid Metabolism	-6.620	0.000	0.299	-2.510	0.284	0.046
glycoursodeoxycholate	Lipid	Secondary Bile Acid Metabolism	3.747	0.054	0.102	2.148	0.237	0.056
tauroursodeoxycholate	Lipid	Secondary Bile Acid Metabolism	5.633	0.000	0.488	4.032	0.030	0.174
glycocholenate sulfate*	Lipid	Secondary Bile Acid Metabolism	10.060	0.004	0.219	0.101	0.981	0.000
taurocholenate sulfate	Lipid	Secondary Bile Acid Metabolism	7.321	0.000	0.472	3.683	0.306	0.042
glycodeoxycholate sulfate	Lipid	Secondary Bile Acid Metabolism	-0.814	0.691	0.005	-1.190	0.491	0.019

Severity prediction model with significant metabolite

Figure 3: significant metabolites in M to S alcoholic hepatitis comparison